Enhanced zein reduction in transgenic corn seed

ABSTRACT

Anti-sense-oriented RNA gene suppression agents in the form of a loop of anti-sense-oriented RNA is produced in cells of transgenic organisms, e.g. plants, by transcription from a recombinant DNA construct that comprises in 5′ to 3′ order a promoter element operably linked to more than one anti-sense-oriented DNA element and one or more complementary DNA elements.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority under 35 U.S.C. 119(e) to provisionalapplications Ser. No. 60/543,157, filed Feb. 10, 2004, No. 60/543,187,filed Feb. 10, 2004 and No. 60/600,859, filed Aug. 11, 2004; and toutility application Ser. No. 11/057062, filed Feb. 10, 2005, thedisclosures of all of which are incorporated herein by reference intheir entireties.

INCORPORATION OF SEQUENCE LISTING

A computer readable form of the sequence listing is contained in thefile named “53428B.ST25.txt” which is 21 kb (measured in MS-Windows) andwas created on Feb. 9, 2005 and is located on a CDROM, which is filedherewith and herein incorporated by reference.

FIELD OF THE INVENTION

Disclosed herein are seeds for transgenic corn having elevated aminoacid levels, recombinant DNA constructs for producing gene-suppressingloops of anti-sense RNA and methods of making and using such constructsand transgenic plants expressing gene-suppressing loops of anti-senseRNA.

BACKGROUND

Certain plants have low levels of specific amino acids compared to otherplants, or compared to hypothetical nutritionally “perfect” proteinmodels based on milk or egg. By these standards, corn has low levels oflysine, methionine and tryptophan. Efforts to increase amino acid levelsin transgenic plants include expressing recombinant DNA which encodesproteins in an amino acid synthesis pathway at higher levels than nativegenes. One such gene for producing enhanced levels of lysine in corn isa bacterial dihydropicolinic [is this correct?]acid synthase. A strategyfor achieving even higher levels of amino acids includes suppression ofgenes encoding proteins in amino acid catabolic pathways.

Gene suppression includes any of the well-known methods for suppressingtranscription of a gene or the accumulation of the mRNA corresponding tothat gene, thereby preventing translation of the transcript intoprotein. More particularly, gene suppression by inserting a recombinantDNA construct with anti-sense oriented DNA to regulate gene expressionin plant cells is disclosed in U.S. Pat. No. 5,107,065 (Shewmaker etal.) and U.S. Pat. No. 5,759,829 (Shewmaker et al.). Plants transformedusing such anti-sense oriented DNA constructs for gene suppression cancomprise integrated DNA arranged as an inverted repeat from co-insertionof several copies of the transfer DNA (T-DNA) into plants byAgrobacterium-mediated transformation, as disclosed by Redenbaugh et al.in “Safety Assessment of Genetically Engineered Flavr Savr™ Tomato, CRCPress, Inc. (1992). Inverted repeat insertions can make up a part or allof the T-DNA, e.g. the T-DNA can contain an inverted repeat of acomplete or partial anti-sense construct. Screening for inserted DNAcomprising inverted repeat elements can improve the efficiency ofidentifying transformation events effective for gene silencing when thetransformation construct is a simple anti-sense DNA construct.

Gene suppression caused by an inserted recombinant DNA construct withsense-oriented DNA is disclosed in U.S. Pat. No. 5,283,184 (Jorgensen etal.) and U.S. Pat. No. 5,231,020 (Jorgensen et al.). Inserted T-DNAproviding gene suppression in plants transformed with such senseconstructs by Agrobacterium is organized predominantly in invertedrepeat structures, as disclosed by Jorgensen et al., Mol. Gen. Genet.,207: 471-477 (1987). See also Stam et al., The Plant Journal, 12: 63-82(1997) and De Buck et al., Plant Mol. Biol. 46 433-445 (2001), who usedsegregation studies to support Jorgensen's finding that in many eventsgene silencing is mediated by multimeric transgene T-DNA where theT-DNAs are arranged in inverted repeats. Screening for inserted DNAcomprising inverted repeat elements can improve the gene silencingefficiency when transforming with simple sense-orientated DNAconstructs.

Gene silencing can also be effected by transcribing RNA from both asense and an anti-sense oriented DNA using two separate transcriptionunits, e.g. as disclosed by Shewmaker et al. in U.S. Pat. No. 5,107,065where in Example 1 a binary vector was prepared with both sense andanti-sense aroA genes. Similar constructs are disclosed in InternationalPublication No. WO 99/53050 (Waterhouse et al.). See also U.S. Pat. No.6,326,193 where gene targeted DNA is operably linked to opposingpromoters.

Gene suppression can be achieved in plants by using transformationconstructs that are capable of generating an RNA that can formdouble-stranded RNA along at least part of its length. Gene suppressionin plants is disclosed in EP 0426195 A1 (Goldbach et al.) whererecombinant DNA constructs for transcription into hairpin RNA providedtransgenic plants with resistance to tobacco spotted wilt virus. Seealso Sijen et al., The Plant Cell, Vol. 8, 2277-2294 (1996) whichdiscloses the use of constructs carrying inverted repeats (sensefollowed by anti-sense) of a cowpea mosaic virus gene in transgenicplants to mediate virus resistance. See also International PublicationNo. 98/53083 (Grierson et al.) and related U.S. Patent ApplicationPublication No. 2003/0175965 A1 (Lowe et al.) which disclose genesuppression using a double stranded RNA construct comprising a genecoding sequence preceded by an inverted repeat of the 5′ UTR. Constructsfor posttranscriptional gene suppression in plants by double-strandedRNA of the target gene are also disclosed in International PublicationNo. WO 99/53050 (Waterhouse et al.) and International Publication No. WO99/49029 (Graham et al.). See also U.S. Patent Application PublicationNo. 2002/0048814 A1 (Oeller) where DNA constructs are transcribed tosense or anti-sense RNA with a hairpin-forming poly(T)-poly(A) tail. Seealso U.S. Patent Application Publication No. 2003/0018993 A1 (Guttersonet al.) where sense or anti-sense DNA is followed by an inverted repeatof the 3′ untranslated region of the NOS gene. See also U.S. PatentApplication Publication No. 2003/0036197 A1 (Glassman et al.) where RNAfor reducing the expression of target mRNA comprises a segment withhomology to target mRNA and a segment with complementary RNA regionsthat are unrelated to endogenous RNA.

The production of dsRNA in plants to inhibit gene expression, e.g. in anematode feeding on the plant, is disclosed U.S. Pat. No. 6,506,559(Fire et al.). Multi-gene suppression vectors for use in plants aredisclosed in U.S. patent application Ser. No. 10/465,800 (Fillatti).

Transcriptional suppression such as promoter trans suppression can beeffected by a expressing a DNA construct comprising a promoter operablylinked to inverted repeats of promoter DNA from a target gene.Constructs useful for such gene suppression mediated by promoter transsuppression are disclosed by Mette et al., The EMBO Journal, Vol. 18,pp. 241-148, (1999) and by Mette et al., The EMBO Journal, Vol. 19, pp.5194-5201-148, (2000), both of which are incorporated herein byreference.

All of the above-described patents, applications and internationalpublications disclosing materials and methods for gene suppression inplants are incorporated herein by reference.

SUMMARY OF THE INVENTION

This invention provides methods and recombinant DNA constructs usefulfor producing anti-sense-oriented RNA for gene suppression in transgenicorganisms. In one aspect of the invention a recombinant DNA constructfor suppressing a plurality of target genes comprises in 5′ to 3′ ordera promoter element operably linked to an anti-sense-oriented DNA elementand a sense-oriented DNA element, in which the sense-oriented DNAelement is shorter than the anti-sense-oriented DNA element andsense-oriented RNA transcribed from the sense-oriented DNA iscomplementary to the 5′-most end of anti-sense-oriented RNA transcribedfrom the anti-sense-oriented DNA element, whereby the transcribed RNAforms a into a loop of anti-sense-oriented RNA for suppressing theplurality of target genes.

The sense-oriented DNA can be cloned as an inverted repeat of a 5′-mostsegment of the anti-sense-oriented DNA element. Constructs with suchsense-oriented DNA are transcribed to RNA that forms a loop ofanti-sense-oriented RNA closed at its ends with a double-stranded RNA(dsRNA) segment, e.g. as illustrated in FIG. 1. To form ananti-sense-oriented RNA loop the complementary DNA element isconveniently not more than about one-half the length of theanti-sense-oriented DNA element, and preferably not more than one-thirdthe length of the anti-sense-oriented DNA element, e.g. not more thanone-quarter the length of the anti-sense-oriented DNA element. Theoverall lengths of the combined DNA elements can vary. For instance, theanti-sense-oriented DNA element can consist of from 500 to 5000nucleotides and the complementary DNA element can consist of from 50 to500 nucleotides. In many cases it will be useful for theanti-sense-oriented DNA segment to be more than twice the length of thesense-oriented DNA segment to allow for formation of ananti-sense-oriented RNA loop.

The anti-sense transcription unit can be designed to suppress multiplegenes where the DNA is arranged with two or more anti-sense-orientedelements from different genes targeted for suppression followed by acomplementary sense-oriented element, e.g. complementary to at least apart of the 5′most anti-sense element.

This invention also provides methods of suppressing the expression of agene by providing in the cells of a plant a recombinant DNA construct ofthis invention that transcribes to an anti-sense loop of RNA. In otheraspects of the invention, e.g. for providing traits other than plantswith enhanced amino acid, the gene targeted for suppression can be aplant gene, a plant pest gene, a plant pathogen gene or a combinationthereof. In the constructs, methods and plants of this invention thegene targeted for silencing can be a native gene or an exogenous gene ora gene in an organism that ingests or contacts the tissues of the plantthat have cells comprising anti-sense RNA in a loop according to thisinvention. Plant pathogens include viruses such as cucumber mosaicvirus, bacteria such as Erwinia stewartii (Stewart's wilt of corn) andfungi such as Phakopsora pachyrhizi (soybean rust fungus); plant pestsinclude nematodes such as soybean cyst nematode and root knot nematode,and insects of various orders including Lepidoptera (e.g., European cornborer), Coleoptera (e.g., spotted cucumber beetle) and Homoptera (e.g.,aphids).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic illustration of a recombinant DNA construct usefulin this invention to produce an anti-sense-oriented loop of RNA.

FIG. 2 is a Western analysis indicating gene suppression using aconstruct of this invention.

FIG. 3 shows mass spectroscopy spectra indicating zein content in seeds.

FIG. 4 is a graphic illustration of the design of the vector constructsused in an aspect of this invention.

FIG. 5 shows mass spectroscopy spectra indicating zein content in wildtype and transgenic seeds.

FIG. 6 illustrates the correlation between the protein content of thekernels and their levels of free amino acids.

DETAILED DESCRIPTION

SEQ ID NO:1 and SEQ ID NO:2 are nucleotide sequences of recombinant DNAconstructs useful for transcribing RNA that can form ananti-sense-oriented RNA loop for suppressing one or multiple genes intransgenic plants. See Tables 1 and 2 for a description of elements ofthose constructs.

As used herein, “complementary” refers to polynucleotides that arecapable of hybridizing, e.g. sense and anti-sense strands of DNA orself-complementary strands of RNA, due to complementarity of alignednucleotides permitting C-G and A-T or A-U bonding.

As used herein “vector” means a DNA molecule capable of replication in ahost cell and/or to which another DNA segment can be operatively linkedso as to bring about replication of the attached segment. A plasmid isan exemplary vector.

As used herein a “transgenic” organism, e.g. plant or seed, is one whosegenome has been altered by the incorporation of recombinant DNAcomprising exogenous genetic material or additional copies of nativegenetic material, e.g. by transformation or recombination of theorganism or an ancestral organism. Transgenic plants include progenyplants of an original plant derived from a transformation processincluding progeny of breeding transgenic plants with wild type plants orother transgenic plants. Crop plants of particular interest in thepresent invention include, but are not limited to maize, soybean,cotton, canola (rape), wheat, rice, sunflower, safflower and flax. Othercrops of interest include plants producing vegetables, fruit, grass andwood.

Recombinant DNA Constructs for Plant Transformation

Recombinant DNA constructs for producing looped, anti-sense RNA, genesuppression agents in transgenic plants can be readily prepared by thoseskilled in the art. Typically, such a DNA construct comprises as aminimum a promoter active in the tissue targeted for suppression, atranscribable DNA element having a sequence that is complementary tonucleotide sequence of a gene targeted for suppression and atranscription terminator element. The targeted gene element copied foruse in transcribable DNA in the gene suppression construct can be apromoter element, an intron element, an exon element, a 5′ UTR element,or a 3′UTR element. Although the minimum size of DNA copied fromsequence of a gene targeted for suppression is believed to be about 21or 23 nucleotides; larger nucleotide segments are preferred, e.g. up thefull length of a targeted gene. Useful lengths of either DNA segment arein the range of 50 to 5000 nucleotides, say anti-sense-oriented DNA of500 to 5000 nucleotides in length and complementary DNA elements can be50 to 500 or more nucleotides in length. The DNA element can comprisemultiple parts of a gene, e.g. nucleotides that are complementary tocontiguous or separated gene elements of UTR, exon and intron. Suchconstructs may also comprise other regulatory elements, DNA encodingtransit peptides, signal peptides, selective markers and screenablemarkers as desired.

With reference to FIG. 1 there is schematically shown a recombinant DNAconstruct comprising a promoter element, an anti-sense-oriented DNAelement (denoted “a/s DNA”), a complementary sense-oriented DNA element(denoted “s DNA”) and DNA providing polyadenylation signals and site(denoted “polyA site”). The DNA construct is transcribed to RNAcomprising an anti-sense-oriented RNA segment and a complementary RNAsegment that is complementary to the 5′-most end of theanti-sense-oriented RNA segment. The 5′ and 3′ ends of the anti-senseRNA can self hybridize to form a double-stranded RNA segment that closesa loop of anti-sense-oriented RNA. For example, if the nucleotidesequence of the 5′-most end of the strand of transcribedanti-sense-oriented DNA is 5′-CGGCATA---, the sequence of the 3′-mostend of the transcribed strand of the inverted repeat DNA will be---TATGCCG-3′ which is readily cloned from the source DNA providing theanti-sense element. With such sequences the loop of anti-sense-orientedRNA will extend from one side of a dsRNA segment, e.g.5′-GCCGUAU-------- 3′-CGGCAUA--------

The anti-sense-oriented DNA and its self-complementary DNA can becontiguous or separated by vector DNA, e.g. up to about 100 nucleotidesor so of vector DNA separating restriction sites used for vectorassembly.

Recombinant DNA constructs can be assembled using commercially availablematerials and methods known to those of ordinary skill in the art. Auseful technology for building DNA constructs and vectors fortransformation is the GATEWAY™ cloning technology (available fromInvitrogen Life Technologies, Carlsbad, Calif.) uses the site specificrecombinase LR cloning reaction of the Integrase att system frombacterophage lambda vector construction, instead of restrictionendonucleases and ligases. The LR cloning reaction is disclosed in U.S.Pat. Nos. 5,888,732 and 6,277,608, U.S. Patent Application Publications2001283529, 2001282319 and 20020007051, all of which are incorporatedherein by reference. The GATEWAY™ Cloning Technology Instruction Manualthat is also supplied by Invitrogen also provides concise directions forroutine cloning of any desired DNA into a vector comprising operableplant expression elements.

An alternative vector fabrication method employs ligation-independentcloning as disclosed by Aslanidis, C. et al., Nucleic Acids Res., 18,6069-6074, 1990 and Rashtchian, A. et al., Biochem., 206, 91-97, 1992where a DNA fragment with single-stranded 5′ and 3′ ends are ligatedinto a desired vector that can then be amplified in vivo.

Numerous promoters that are active in plant cells have been described inthe literature. These include promoters present in plant genomes as wellas promoters from other sources, including nopaline synthase (nos)promoter and octopine synthase (ocs) promoters carried on tumor-inducingplasmids of Agrobacterium tumefaciens, caulimovirus promoters such asthe cauliflower mosaic virus or figwort mosaic virus promoters. Forinstance, see U.S. Pat. Nos. 5,322,938 and 5,858,742 which discloseversions of the constitutive promoter derived from cauliflower mosaicvirus (CaMV35S), U.S. Pat. No. 5,378,619 which discloses a FigwortMosaic Virus (FMV) 35S promoter, U.S. Pat. No. 5,420,034 which disclosesa napin promoter, U.S. Pat. No. 6,437,217 which discloses a maize RS81promoter, U.S. Pat. No. 5,641,876 which discloses a rice actin promoter,U.S. Pat. No. 6,426,446 which discloses a maize RS324 promoter, U.S.Pat. No. 6,429,362 which discloses a maize PR-1 promoter, U.S. Pat. No.6,232,526 which discloses a maize A3 promoter, U.S. Pat. No. 6,177,611which discloses constitutive maize promoters, U.S. Pat. No. 6,433,252which discloses a maize L3 oleosin promoter, U.S. Pat. No. 6,429,357which discloses a rice actin 2 promoter and intron, U.S. Pat. No.5,837,848 which discloses a root specific promoter, U.S. Pat. No.6,084,089 which discloses cold inducible promoters, U.S. Pat. No.6,294,714 which discloses light inducible promoters, U.S. Pat. No.6,140,078 which discloses salt inducible promoters, U.S. Pat. No.6,252,138 which discloses pathogen inducible promoters, U.S. Pat. No.6,175,060 which discloses phosphorus deficiency inducible promoters,U.S. Pat. No. 6,635,806 which discloses a coixin promoter, U.S.2002/0192813A1 which discloses 5′, 3′ and intron elements useful in thedesign of effective plant expression vectors, U.S. 2004/0216189 A1 whichdiscloses a maize chloroplast aldolase promoter, and U.S. 2004/0123347A1which discloses water-deficit inducible promoters, all of which areincorporated herein by reference. These and numerous other promotersthat function in plant cells are known to those skilled in the art andavailable for use in recombinant polynucleotides of the presentinvention to provide for expression of desired genes in transgenic plantcells.

Furthermore, the promoters may be altered to contain multiple “enhancersequences” to assist in elevating gene expression. Such enhancers areknown in the art. By including an enhancer sequence with suchconstructs, the expression of the selected protein may be enhanced.These enhancers often are found 5′ to the start of transcription in apromoter that functions in eukaryotic cells, but can often be insertedupstream (5′) or downstream (3′) to the coding sequence. In someinstances, these 5′ enhancing elements are introns. Particularly usefulas enhancers are the 5′ introns of the rice actin 1 (see U.S. Pat. No.5,641,876) and rice actin 2 genes, the maize alcohol dehydrogenase geneintron, the maize heat shock protein 70 gene intron (U.S. Pat. No.5,593,874) and the maize shrunken 1 gene.

In other aspects of the invention, sufficient expression in plant seedtissues is desired to effect improvements in seed composition. Exemplarypromoters for use for seed composition modification include promotersfrom seed genes such as napin (U.S. Pat. No. 5,420,034), maize L3oleosin (U.S. Pat. No. 6,433,252), zein Z27 (Russell et al. (1997)Transgenic Res. 6(2):157-166), globulin 1 (Belanger et al (1991)Genetics 129:863-872), glutelin 1 (Russell (1997) supra), andperoxiredoxin antioxidant (Per1) (Stacy et al. (1996) Plant Mol Biol.31(6):1205-1216).

Recombinant DNA constructs prepared in accordance with the inventionwill often include a 3′ element that typically contains apolyadenylation signal and site, especially if the recombinant DNA isintended for protein expression as well as gene suppression. Well-known3′ elements include those from Agrobacterium tumefaciens genes such asnos 3′, tml 3′, tmr 3′, tms 3′, ocs 3′, tr7 3′, e.g. disclosed in U.S.Pat. No. 6,090,627, incorporated herein by reference; 3′ elements fromplant genes such as wheat (Triticum aesevitum) heat shock protein 17(Hsp17 3′), a wheat ubiquitin gene, a wheat fructose-1,6-biphosphatasegene, a rice glutelin gene a rice lactate dehydrogenase gene and a ricebeta-tubulin gene, all of which are disclosed in U.S. published patentapplication 2002/0192813 A1, incorporated herein by reference; and thepea (Pisum sativum) ribulose biphosphate carboxylase gene (rbs 3′), and3′ elements from the genes within the host plant.

The gene-suppressing recombinant DNA constructs can also be stacked withDNA imparting other traits of agronomic interest including DNA providingherbicide resistance or insect resistance such as using a gene fromBacillus thuringensis to provide resistance against lepidopteran,coliopteran, homopteran, hemiopteran, and other insects. Herbicides forwhich resistance is useful in a plant include glyphosate herbicides,phosphinothricin herbicides, oxynil herbicides, imidazolinoneherbicides, dinitroaniline herbicides, pyridine herbicides, sulfonylureaherbicides, bialaphos herbicides, sulfonamide herbicides and glufosinateherbicides. Persons of ordinary skill in the art are enabled inproviding stacked traits by reference to U.S. patent applicationpublications 2003/0106096A1 and 2002/0112260A1 and U.S. Pat. Nos.5,034,322; 5,776,760; 6,107,549 and 6,376,754 and toinsect/nematode/virus resistance by reference to U.S. Pat. Nos.5,250,515; 5,880,275; 6,506,599; 5,986,175 and U.S. Patent ApplicationPublication 2003/0150017 A1, all of which are incorporated herein byreference.

Transformation Methods—Numerous methods for transforming plant cellswith recombinant DNA are known in the art and may be used in the presentinvention. Two commonly used methods for plant transformation areAgrobacterium-mediated transformation and microprojectile bombardment.Microprojectile bombardment methods are illustrated in U.S. Pat. Nos.5,015,580 (soybean); 5,550,318 (corn); 5,538,880 (corn); 5,914,451(soybean); 6,160,208 (corn); 6,399,861 (corn) and 6,153,812 (wheat) andAgrobacterium-mediated transformation is described in U.S. Pat. Nos.5,159,135 (cotton); 5,824,877 (soybean); 5,591,616 (corn); and 6,384,301(soybean), all of which are incorporated herein by reference. ForAgrobacterium tumefaciens based plant transformation system, additionalelements present on transformation constructs will include T-DNA leftand right border sequences to facilitate incorporation of therecombinant polynucleotide into the plant genome.

In general it is useful to introduce recombinant DNA randomly, i.e. at anon-specific location, in the genome of a target plant line. In specialcases it may be useful to target recombinant DNA insertion in order toachieve site-specific integration, e.g. to replace an existing gene inthe genome, to use an existing promoter in the plant genome, or toinsert a recombinant polynucleotide at a predetermined site known to beactive for gene expression. Several site specific recombination systemsexist that are known to function implants include cre-lox as disclosedin U.S. Pat. No. 4,959,317 and FLP-FRT as disclosed in U.S. Pat. No.5,527,695, both incorporated herein by reference.

Transformation methods of this invention are preferably practiced intissue culture on media and in a controlled environment. “Media” refersto the numerous nutrient mixtures that are used to grow cells in vitro,that is, outside of the intact living organism. Recipient cell targetsinclude, but are not limited to, meristem cells, callus, immatureembryos and gametic cells such as microspores, pollen, sperm and eggcells. It is contemplated that any cell from which a fertile plant maybe regenerated is useful as a recipient cell. Callus may be initiatedfrom tissue sources including, but not limited to, immature embryos,seedling apical meristems, microspores and the like. Cells capable ofproliferating as callus are also recipient cells for genetictransformation. Practical transformation methods and materials formaking transgenic plants of this invention, e.g. various media andrecipient target cells, transformation of immature embryos andsubsequent regeneration of fertile transgenic plants are disclosed inU.S. Pat. Nos. 6,194,636 and 6,232,526, which are incorporated herein byreference.

The seeds of transgenic plants can be harvested from fertile transgenicplants and be used to grow progeny generations of transformed plants ofthis invention including hybrid plants line for screening of plantshaving an enhanced agronomic trait. In addition to direct transformationof a plant with a recombinant DNA, transgenic plants can be prepared bycrossing a first plant having a recombinant DNA with a second plantlacking the DNA. For example, recombinant DNA can be introduced intofirst plant line that is amenable to transformation to produce atransgenic plant that can be crossed with a second plant line tointrogress the recombinant DNA into the second plant line. A transgenicplant with recombinant DNA providing an enhanced agronomic trait, e.g.enhanced yield, can be crossed with transgenic plant line having otherrecombinant DNA that confers another trait, e.g. herbicide resistance orpest resistance, to produce progeny plants having recombinant DNA thatconfers both traits. Typically, in such breeding for combining traitsthe transgenic plant donating the additional trait is a male line andthe transgenic plant carrying the base traits is the female line. Theprogeny of this cross will segregate such that some of the plants willcarry the DNA for both parental traits and some will carry DNA for oneparental trait; such plants can be identified by markers associated withparental recombinant DNA Progeny plants carrying DNA for both parentaltraits can be crossed back into the female parent line multiple times,e.g. usually 6 to 8 generations, to produce a progeny plant withsubstantially the same genotype as one original transgenic parental linebut for the recombinant DNA of the other transgenic parental line

In the practice of transformation DNA is typically introduced into onlya small percentage of target cells in any one transformation experiment.Marker genes are used to provide an efficient system for identificationof those cells that are stably transformed by receiving and integratinga transgenic DNA construct into their genomes. Preferred marker genesprovide selective markers that confer resistance to a selective agent,such as an antibiotic or herbicide. Any of the herbicides to whichplants of this invention may be resistant are useful agents forselective markers. Potentially transformed cells are exposed to theselective agent. In the population of surviving cells will be thosecells where, generally, the resistance-conferring gene is integrated andexpressed at sufficient levels to permit cell survival. Cells may betested further to confirm stable integration of the exogenous DNA.Commonly used selective marker genes include those conferring resistanceto antibiotics such as kanamycin and paromomycin (nptII), hygromycin B(aph IV) and gentamycin (aac3 and aacC4) or resistance to herbicidessuch as glufosinate (bar or pat) and glyphosate (aroA or EPSPS).Examples of such selectable are illustrated in U.S. Pat. Nos. 5,550,318;5,633,435; 5,780,708 and 6,118,047, all of which are incorporated hereinby reference. Screenable markers that provide an ability to visuallyidentify transformants can also be employed, e.g., a gene expressing acolored or fluorescent protein such as a luciferase or green fluorescentprotein (GFP) or a gene expressing a beta-glucuronidase or uidA gene(GUS) for which various chromogenic substrates are known.

Cells that survive exposure to the selective agent, or cells that havebeen scored positive in a screening assay, may be cultured inregeneration media and allowed to mature into plants. Developingplantlets can be transferred to plant growth mix, and hardened off,e.g., in an environmentally controlled chamber at about 85% relativehumidity, 600 ppm CO₂, and 25-250 microeinsteins m⁻² s⁻¹ of light, priorto transfer to a greenhouse or growth chamber for maturation. Plants areregenerated from about 6 weeks to 10 months after a transformant isidentified, depending on the initial tissue. Plants may be pollinatedusing conventional plant breeding methods known to those of skill in theart and seed produced, e.g. self-pollination is commonly used withtransgenic corn. The regenerated transformed plant or its progeny seedor plants can be tested for expression of the recombinant DNA andscreened for the presence of enhanced agronomic trait.

Transgenic Plants and Seeds

Transgenic plant seed provided by this invention are grown to generatetransgenic plants having an enhanced trait as compared to a controlplant. Such seed for plants with enhanced agronomic trait is identifiedby screening transformed plants or progeny seed for enhanced trait. Forefficiency a screening program is designed to evaluate multipletransgenic plants (events) comprising the recombinant DNA, e.g. multipleplants from 2 to 20 or more transgenic events.

Transgenic plants grown from transgenic seed provided herein demonstrateimproved agronomic traits that contribute to increased yield or othertrait that provides increased plant value, including, for example,improved seed quality. Of particular interest are plants having enhancedyield resulting from improved plant growth and development, stresstolerance, improved seed development, higher light response, improvedflower development, or improved carbon and/or nitrogen metabolism

Many transgenic events that survive to fertile transgenic plants thatproduce seeds and progeny plants will not exhibit an enhanced agronomictrait. Screening is necessary to identify the transgenic plant havingenhanced agronomic traits from populations of plants transformed asdescribed herein by evaluating the trait in a variety of assays todetect an enhanced agronomic trait. These assays also may take manyforms, including but not limited to, analyses to detect changes in thechemical composition, biomass, physiological properties, morphology ofthe plant.

The following examples illustrate aspects of the invention.

EXAMPLE 1

This example illustrates preparation of a transformation vector usefulfor inserting a recombinant DNA construct of this invention into atransgenic plant to practice a method of this invention.

The LKR/SDH gene encodes a pre-protein for lysine ketoglutaratereductase (LKR) and saccharopine dehydrogenase (SDH) which are enzymesin a lysine catabolic pathway. Suppression of LKR is manifest inmodification, e.g. increase, of lysine content. Suppression of LKR iseffected by expressing in a plant a recombinant DNA construct thatproduces a stabilized anti-sense RNA transcribed fromanti-sense-oriented LKR DNA and sense-oriented LKR DNA that forms a loopof anti-sense-oriented RNA.

A transformation vector is prepared comprising two transcription unitsbetween right and left borders from Agrobacterium tumefaciens. Onetranscription unit for a marker comprised:

-   -   (a) DNA of a rice actin promoter and rice actin intron,    -   (b) DNA of a chloroplast transit peptide from Arabidopsis EPSPS    -   (c) DNA of A. tumefaciens aroA (a glyphosate-resistant marker),        and    -   (d) DNA of A. tumefaciens NOS terminator,        The other transcription unit for LKR gene suppression comprised:    -   (a) DNA of Zea mays GLB1 promoter,    -   (b) DNA of a Zea mays ADH1 intron,    -   (c) Anti-sense-oriented DNA fragment of Zea mays LKR,    -   (d) Sense-oriented DNA fragment of Zea mays LKR, and    -   (e) DNA of Zea mays GLB1 terminator.

SEQ ID NO: 1 is DNA sequence of a transformation vector comprising theabove-described marker and gene suppression elements. See Table 1 belowfor a description of the elements of the transformation vector containedwithin SEQ ID NO:1. TABLE 1 Bases of SEQ ID NO: 1 Description of DNAsegment  1-357 A. tumefaciens right border  376-1774 DNA of a rice actinpromoter and rice actin intron 1784-2011 DNA of A. tumefaciens EPSPSchloroplast transit peptide 2012-3379 DNA of A. tumefaciens aroA(glyphosate-resistant marker) 3395-3647 DNA of A. tumefaciens NOSterminator 3691-4686 DNA of Zea mays Glb1 terminator 4692-5145Sense-oriented DNA element from Zea mays LKR 5152-6118Anti-sense-oriented DNA element from Zea mays LKR 6123-6680 DNA of a Zeamays ADH1 intron 6687-8082 DNA of Zea mays GLB1 promoter 8149-8590 A.tumefaciens left border

A vector prepared with the elements listed in Table 1 was used totransform corn plant tissue. Transgenic corn plants were obtained byAgrobacterium-mediated transformation. Transgenic plants from twoseparate transgenic insertion events were grown to produce F1 seed. Sixmature seeds from each event were analyzed to determine success oftransformation and suppression of LKR. The mature transgenic seeds weredissected to extract protein that was analyzed by Western analysis. Withreference to FIG. 2, seed from one of the events showed no reduction inLKR as compared to wild type; and seed from the other event was shown tobe segregating (1:1 hemizygous:wild type) as three of the six seedsshowed substantial reduction in LKR as compared to wild type.

EXAMPLE 2

This example illustrates a wide scope of embodiments of transformationvectors useful for inserting a recombinant DNA construct of thisinvention into a transgenic plant to practice a method of thisinvention. Transformation vectors were prepared using the following DNAelements where:

-   -   (a) “pGcx” refers to DNA for a promoter derived from a gamma        coixin gene from Coix lacryma-jobi;    -   (b) “pZ27” refers to DNA for a promoter derived from a gamma        zein gene from Zea mays;    -   (c) “pZ27t” refers to DNA for a truncated promoter having 59        nucleotides leader sequence deleted from the 3′ region of pZ27;    -   (d) “Z19as” refers to DNA for an antisense-oriented segment of        351 nucleotides from the coding sequence of a 19 kilo dalton        alpha zein gene from Zea mays;    -   (e) “Z19s” refers to DNA for a sense-oriented segment of 351        nucleotides from the coding sequence of a 19 kilo dalton alpha        zein gene from Zea mays, which is an inverted repeat of Z19as;    -   (f) “Z22as” refers to DNA for an antisense-oriented segment of        789 nucleotides from the coding sequence of a 22 kilo dalton        alpha zein gene from Zea mays;    -   (g) “Z22asL” refers to DNA for an antisense-oriented segment of        785 nucleotides from the coding sequence of a 22 kilo dalton        alpha zein gene from Zea mays;    -   (h) “Z22asSI” refers to DNA for an antisense-oriented segment of        789 nucleotides from the coding sequence of a 22 kilo dalton        alpha zein gene from Zea mays having a 520 nucleotide long        spliceable intron from a GB1 gene intron 3 from Zea mays        inserted in the unpaired region;    -   (i) “Z22s” refers to DNA for a sense-oriented segment of 289        nucleotides from the coding sequence of a 22 kilo dalton alpha        zein gene from Zea mays, which is an inverted repeat of the 5′        end of Z22as; and

(j) “TE9” refers to DNA for a sense oriented polyadenylation signal andsite element from an RbcS2 gene from Pisum sativum.

With reference to Table 2 and SEQ ID NO:2 a transformation vectorcomprising “construct 2a” was made in the manner of Example 1 exceptthat the transcription unit for LKR gene suppression was replaced by atranscription unit comprising the elements illustrated in the followingschematic:

“Construct 2a” pZ27 - Z19as - Z22asL - Z22s - Z19s - TE9 TABLE 2 Basesof SEQ ID NO: 2 description of DNA segment  1-357 A. tumefaciens rightborder  376-1774 DNA of a rice actin promoter and rice actin intron1784-2011 DNA of A. tumefaciens EPSPS chloroplast transit peptide2012-3379 DNA of A. tumefaciens aroA (glyphosate-resistant marker)3395-3647 DNA of A. tumefaciens NOS terminator 3479-4391 DNA of Pisumsativum RbcS2 terminator 4398-4748 DNA for Z19s 4755-5043 DNA for Z22s5050-5835 DNA of Z22asL 5842-6192 DNA of Z19as 6204-7305 DNA of Zea maysZ27 promoter 7353-7794 A. tumefaciens left border

Corn callus was transformed and events with a single copy of thetransformation vector were selected for growth into plants. Seed fromplants grown from 26 of 29 single copy events showed substantialreduction of the 19 kilo dalton alpha zeins and the 22 kilo Dalton alphazeins.

Other transformation vectors were made in a similar manner using theelements illustrated in the following Table 3. TABLE 3 Construct 2b1pGcx-Z19as-Z22asSI-Z22s-Z19s-TE9 Construct 2b2*pGcx-Z19as-Z22asSI-Z22s-Z19s-TE9 Construct 2cpZ27-Z19as-Z22asSI-Z22s-Z19s-TE9 Construct 2dPZ27t-Z19as-Z22asSI-Z22s-Z19s-TE9 Construct 2ePZ27-Z19as-Z22asL-Z19s-TE9*construct 2b2 was inserted into a transformation vector that alsoincluded a transcription unit for expressing another gene having apromoter contiguous to pGcx.

The efficiency of suppressing the alpha zeins in seeds produced byplants grown from single copy events is reported in Table 4 whichreports the number of transgenic events with reduction of zeins ascompared to the total number of transgenic events generated in eachconstruct tested. The zein reduction phenotype is observed by MALDI-TOFMS (Matrix-Assisted-Laser-Desorption Ionization Time-Of-Flight MassSpectrometry) analysis. FIG. 3 is illustrates typical spectra evidencingzein reduction. TABLE 4 Construct 19 kD zein 19 and 22 kD zein 2a 26/2926/29  2b1  0/21  0/21  2b2 5/7 0/7 2c 20/21 18/21 2d 7/8 1/8 2e 12/14 2/14

EXAMPLE 3

This example illustrates zein reduction in transgenic plants harboringconstruct 2a (pMON73566; Table 2 and FIG. 4A) and construct 2e(pMON73566; Table 3, Table 5, and FIG. 4B) as well as the oil, protein,and amino acid profiles of bulked kernels derived from plants grown fromsingle copy events as reported in Table 4.

With reference to Table 5 and SEQ ID NO:2 a transformation vectorcomprising “construct 2e” was made in the manner of Example 1 exceptthat the transcription unit for LKR gene suppression was replaced by atranscription unit comprising the elements illustrated in the followingschematic:

“Construct 2e” PZ27 - Z19as - Z22asL - Z19s - TE9 TABLE 5 Bases of SEQID NO: 2 description of DNA segment  1-357 A. tumefaciens right border 376-1774 DNA of a rice actin promoter and rice actin intron 1784-2011DNA of A. tumefaciens EPSPS chloroplast transit peptide 2012-3379 DNA ofA. tumefaciens aroA (glyphosate-resistant marker) 3395-3647 DNA of A.tumefaciens NOS terminator 3479-4391 DNA of Pisum sativum RbcS2terminator 4398-4748 DNA for Z19s 4750-5535 DNA of Z22asL 5542-5892 DNAof Z19as 5904-7003 DNA of Zea mays Z27 promoter 7353-7794 A. tumefaciensleft border

Corn immature embryo was transformed and events with a single copy ofthe transformation vector were selected for growth into plants. Seedfrom plants grown from 2 of 14 single copy events showed substantialreduction of both the 19 kilo Dalton alpha zeins and the 22 kilo Daltonalpha zeins (Table 4).

Zein Reduction

In transgenic plants harboring pMON73567, which contains dsRNA againstboth 19- and 22-kD α-zein sequences, 26 of 29 plants display reductionin both 19- and 22-kD α-zein accumulation (Table 4, construct 2a; FIG.4A; FIG. 5B). Additionally, in transgenic plants harboring pMON73566,which contains dsRNA against only a 19-kD α-zein sequence and which usesthe 22-kD α-zein sequence as the loop, 2 of 14 plants display reductionin both 19- and 22-kD α-zein accumulation (Table 4, construct 2e; FIG.4B; FIG. 5B). Ten other pMON73566 events exhibit mostly 19-kD α-zeinreduction (Table 4, construct 2e; FIG. 4B; FIG. 5C). Two representativeevents were selected from each construct for advancement to the nextgeneration for collection of homozygous ears for compositional analyses.Events M80442 and M82186 containing the pMON73567 construct, and eventsM80780 and M80791 containing the pMON73566 construct were selected.M80442, M82186 and M80791 exhibit both 19- and 22-kD α-zein reduction.M80780, exhibits only 19-kD α-zein reduction.

Oil Content

Four events (M80442, M82186, M80780 and M80791) from 2 zein reductionconstructs, pMON73566 and pMON73567, were grown in the field andzygosity was determined by a molecular assay. Oil was determined forkernels of homozygous transgene-positive ears and control ears by a wetchemistry oil extraction method. About 100 mg of ground sample wasweighed in an 11-ml pressure cell half filled with sand. Additional sandwas added to fill the cell, a filter was placed on the top, and the cellwas capped with a screw cap. The cell was placed on the carousel of theDionex Accelerated Solvent Extractor (Dionex, Sunnyvale, Calif.)following the manufacturer's protocol. The oil was extracted withpetroleum ether at 1000 psig (pounds per square inch gauge) at 105° C.in three extraction steps. The final extraction product was added to apre-weighed vial. The solvent was evaporated at 37° C. for two hoursunder a stream of either nitrogen or air and the vial was then weighed.Analysis of oil content of the samples was done in triplicate. Oilconcentration of control kernels averaged 3.8% (Table 6). Average oilconcentration of the transgene-positive kernels ranged from 4.0% to5.2%. The average oil content of seeds from all 4 transgenic eventsrepresents an increase over the average oil content of seeds from wildtype plants; and the increase in oil content from the M82186 event isconsidered to be statistically significant. TABLE 6 Proximate assay andsize of bulked kernels Wild Type pMON73566 pMON73567 % + SD^(a) LH244M80780 M80791 M80442 M82186 Oil^(b) 3.8 ± 0.3 4.1 ± 0.2 4.4 ± 0.1 4.0 ±0.5  5.2 ± 0.5 Protein 9.6 ± 1.1 8.7 ± 1.0 9.9 ± 1.2 9.3 ± 1.0 10.0 ±0.7  Starch 70.4 ± 0.9  68.7 ± 0.4  67.3 ± 0.3  68.5 ± 0.6  67.8 ± 1.0Moisture 9.0 ± 0.4 10.5 ± 0.2  10.4 ± 0.3  10.5 ± 0.2  10.5 ± 0.7Density 1.31 ± 0.00 1.20 ± 0.01 1.20 ± 0.02  1.20 ± 0.01  1.21 ± 0.01Size^(c) 24.9 ± 1.4  24.3 ± 2.0  24.0 ± 0.9  23.9 ± 1.4  23.2 ± 2.2Table 6. The oil, protein and starch contents are calculated on drymatter base. Except oil, the proximate contents of bulked kernels fromeach ear were determined by near infrared transmission (NIT) analysis.^(b)The oil content was obtained by a wet chemistry method.The numbers in bold are statistically different from the LH244 numbersby Dunnett's test (α = 0.05).^(a)Data are means ± standard deviations.^(c)The sizes were measured in 100 kernel weights in grams.Amino Acid Profile

Transgenic plants transformed with antisense constructs targeting 19-kDα-zeins have exhibited an increased lysine content of up to 35% inmature corn seeds (Huang et al., 2004; Huang et al., 2005). In thetransgenic plants haboring the pMON73567 and pMON73566 constructs thattarget both the 19- and 22-kD α-zeins, the increases in lysine contentare significantly greater. As shown in Table 7, among the eventsanalyzed, the least increase in lysine content, 66% ((4035 ppmtransgenic-2438 ppm wild type)/2438 ppm wild type), is observed inM80780 and the most, 105% ((5003 ppm transgenic-2438 ppm wild type)/2438ppm wild type), is found in M80791. Similarly, the transgenic plantshave much higher tryptophan contents as compared to the wild type, LH244(Table 7). TABLE 7 Total amino acid analysis of ground kernels Wild TypepMON73566 pMON73567 Ave ± SD^(a) LH244 M80780 M80791 M80442 M82186 Ala6687 ± 594 5497 ± 631 6417 ± 855 5862 ± 999 6458 ± 322 Arg 4342 ± 2936060 ± 708  7313 ± 1048  6665 ± 1203 7165 ± 655 Asx 5555 ± 377  8928 ±1651 11977 ± 2034 10253 ± 2803 11143 ± 886  Glx 17788 ± 1623 15873 ±2421 18610 ± 2762 16860 ± 3617 18603 ± 1322 Gly 3400 ± 166 4537 ± 4635377 ± 770 4973 ± 783 5290 ± 410 His 1498 ± 126 2350 ± 234 2253 ± 4412305 ± 518 2470 ± 160 Ile 3265 ± 255 3030 ± 368 3700 ± 678 3373 ± 5873578 ± 261 Leu 11265 ± 1074 7318 ± 788  8327 ± 1106  7718 ± 1264 8270 ±497 Lys 2438 ± 132 4035 ± 574 5003 ± 866 4533 ± 780 4800 ± 443 Phe 3760± 282 3032 ± 295 3637 ± 506 3288 ± 571 3455 ± 260 Ser 4067 ± 364 4235 ±465 4620 ± 425 4355 ± 779 4785 ± 325 Thr 3062 ± 226 3572 ± 406 4047 ±446 3783 ± 676 4130 ± 279 Trp 598 ± 48  877 ± 117 1087 ± 158  940 ± 2011040 ± 96  Tyr 3720 ± 307 3430 ± 414 4093 ± 432 3652 ± 624 4045 ± 270Val 4710 ± 325 5312 ± 598  6553 ± 1068  5977 ± 1030 6293 ± 483 Sum 76155± 5996  78087 ± 10057  93013 ± 12760  84535 ± 16220 91553 ± 6286 Lys %(P)^(b)  2.83 ± 0.23  5.23 ± 0.17  5.62 ± 0.29  5.40 ± 0.37  5.33 ± 0.28Trp % (P)^(b)  0.69 ± 0.05  1.14 ± 0.04  1.22 ± 0.03  1.12 ± 0.11  1.15± 0.05 Leu % (P)^(b) 13.00 ± 0.34  9.51 ± 0.15  9.38 ± 0.12  9.21 ± 0.56 9.20 ± 0.34Table 7. Samples were ground mills of bulked mature kernels ofindividual ears.^(a)Data (ppm) are averages of ears within an event ± standarddeviations.Four homologous ears from each event were measured.^(b)They are expressed as the percentages of protein measured in Table 6without the subtraction of moisture.The numbers in bold are statistically different from the LH244 numbersby Dunnett's test (α = 0.05).Asx, asparagine and Aspartate;Glx, glutamine and glutamate.

Aspartate, asparagine and glutamate are among the free amino acids thatexhibit a significant increase in the seeds of these transgenic plants(Table 8). The increased accumulation of these free amino acids has beenshown to enhance lysine biosynthesis in the presence of CordapA (Huanget al., 2005; Monsanto patent application Ser. No. 11/077089, filed Mar.10, 2005). TABLE 8 Free amino acid analysis of ground kernels Wid TypepMON73566 pMON73567 Ave ± SD^(a) LH244 M80780 M80791 M80442 M82186 Ala 95 ± 39 114 ± 73  194 ± 91 172 ± 103  313 ± 122 Arg  50 ± 24 92 ± 64121 ± 40 110 ± 53  160 ± 38 Asn 232 ± 40 2341 ± 667   3726 ± 1102 2844 ±1184 2995 ± 402 Asp 143 ± 30 692 ± 386 1306 ± 375 1040 ± 479  1323 ± 207Glu 256 ± 33 582 ± 492 1064 ± 472 823 ± 670 1207 ± 119 Gln  85 ± 56 182± 186  270 ± 180 255 ± 264 319 ± 40 Gly 18 ± 6 25 ± 13  37 ± 13 31 ± 1544 ± 7 His 24 ± 4 47 ± 21  79 ± 25 63 ± 27 76 ± 5 Ile 13 ± 5 18 ± 12 25± 8 21 ± 9  36 ± 9 Leu 10 ± 3 15 ± 14  25 ± 11 19 ± 14  34 ± 10 Lys  25± 12 40 ± 26  66 ± 39 57 ± 40  70 ± 11 Phe  37 ± 30 19 ± 12 34 ± 8 25 ±11  40 ± 11 Ser  50 ± 21 69 ± 41 115 ± 50 88 ± 53 176 ± 76 Thr 16 ± 6 46± 41  84 ± 36 60 ± 46 110 ± 29 Trp  8 ± 1 17 ± 5  23 ± 4 21 ± 5  25 ± 1Tyr 34 ± 6 94 ± 52 163 ± 18 120 ± 48  182 ± 52 Val  33 ± 10 46 ± 32  76± 29 62 ± 33 101 ± 26 Sum 1134 ± 259 4447 ± 1962  7417 ± 2334 5819 ±3052 7225 ± 336Table 8. Samples were ground mills of bulked mature kernels ofindividual ears.^(a)Data (ppm) are averages of ears within an event ± standarddeviations.Four homologous ears from each event were measured.The numbers in bold are statistically different from the LH244 numbersby Dunnett's test (α = 0.05).Protein Content

The constitutive expression of asparagine synthetase is thought toincrease the protein content of maize seeds by fueling the flux of freeamino acids from vegetative tissues into developing ears. Not only dothese zein reduction lines have elevated levels of free amino acids(Table 8), but also the amount of protein accumulation is proportionalto the level of free amino acids (FIG. 6). These results suggest apossible synergistic effect of combining zein reduction and theexpression of asparagine synthetase that could result in improving boththe quantity and the quality of the maize proteins.

This invention contemplates the use of anti-sense-oriented DNA elementsand sense-oriented DNA elements from other maize zein proteins,including but not limited to additional members of the 19- and 22-kDα-zeins, 16- and 27-kD γ-zeins, 10-kD δ-zeins, and 15-kD β-zeins, as amethod for suppressing transcription of more than one gene or theaccumulation of the mRNA corresponding to those genes thereby preventingtranslation of the transcript into protein. In particular, thecontemplated α-zeins include all variants in the 19-kD size range andall variants in the 22-kD size range.

All of the materials and methods disclosed and claimed herein can bemade and used without undue experimentation as instructed by the abovedisclosure. Although the materials and methods of this invention havebeen described in terms of preferred embodiments and illustrativeexamples, it will be apparent to those of skill in the art thatvariations may be applied to the materials and methods described hereinwithout departing from the concept, spirit and scope of the invention.All such similar substitutes and modifications apparent to those skilledin the art are deemed to be within the spirit, scope and concept of theinvention as defined by the appended claims.

1. A recombinant DNA construct for suppression of a plurality of maizezein target genes, comprising in 5′ to 3′ order a promoter elementoperably linked to a plurality of anti-sense-oriented DNA elements froma plurality of maize zein target genes, the anti-sense-oriented DNAelements designated in 5′ to 3′ order element 1, element 2, and aplurality of sense-oriented DNA elements from a plurality of maize zeintarget genes, designed in 5′ to 3′ order element 3, element 4, whereinthe anti-sense-oriented DNA element 2 has at least a portion of itssequence at its 3′ end that is not complementary either to itself or tothe sense-oriented DNA element 3, and wherein sense-oriented RNAtranscribed by the sense-oriented DNA elements is complementary to the5′-most end of anti-sense-oriented RNA transcribed by theanti-sense-oriented DNA elements.
 2. A recombinant DNA construct ofclaim 1 wherein the anti-sense-oriented DNA elements 1 and 2 are fromtwo different genes targeted for suppression and the sense-oriented DNAelements 3 and 4 are from two different genes targeted for suppression.3. A recombinant DNA construct of claim 1 wherein theanti-sense-oriented DNA element 1 is from a 19-kD α-zein gene targetedfor suppression, the anti-sense-oriented DNA element 2 is from a 22-kDα-zein gene targeted for suppression, the sense-oriented DNA element 3is from a 22-kD α-zein gene targeted for suppression and is shorted thanthe anti-sense-oriented DNA element 2 and is complementary to only the5′ end of element 2 and the sense-oriented DNA element 4 is from a 19-kDα-zein gene targeted for suppression and is complementary to at least aportion of the 5′ end of element
 1. 4. A recombinant DNA construct ofclaim 1 wherein the more than one maize zein target gene is selectedfrom the group consisting of 19- and 22-kD α-zeins, 16- and 27-kDγ-zeins, 10-kD δ-zeins, and 15-kD β-zeins.
 5. A recombinant DNAconstruct for suppression of more than one maize zein target gene thatcomprises in 5′ to 3′ order a promoter element operably linked to morethan one anti-sense-oriented DNA element from more than one maize zeintarget gene, the anti-sense-oriented DNA elements designed in 5′ to 3′order element 1, element 2, and at least one sense-oriented DNA elementfrom at least one maize zein target gene, designated element 3, whereinthe anti-sense-oriented DNA element 2 has at least a portion of itssequence at its 3′ end that is not complementary either to itself or tothe sense-oriented DNA element 3, and sense-oriented RNA transcribed bythe sense-oriented DNA element is complementary to the 5′-most end ofanti-sense-oriented RNA transcribed by the anti-sense-oriented DNAelement(s).
 6. A recombinant DNA construct of claim 5 wherein theanti-sense-oriented DNA elements 1 and 2 are from two genes targeted forsuppression and the sense-oriented DNA element 3 is from a gene targetedfor suppression.
 7. A recombinant DNA construct of claim 5 wherein theanti-sense-oriented DNA element 1 is from a 19-kD α-zein gene targetedfor suppression, the anti-sense-oriented DNA element 2 is from a 22-kDα-zein gene targeted for suppression, and the sense-oriented DNA element3 is from a 19-kD α-zein gene targeted for suppression.
 8. A recombinantDNA construct of claim 5 wherein the more than one maize zein targetgene is selected from the group consisting of 19- and 22-kD α-zeins, 16-and 27-kD γ-zeins, 10-kD δ-zeins, and 15-kD β-zeins and wherein the atleast one maize zein target gene is selected from the group consistingof 19- and 22-kD α-zeins, 16- and 27-kD γ-zeins, 10-kD δ-zeins, and15-kD βzeins.
 9. A method for generating corn seeds having at least oneof enhanced lysine, enhanced oil and enhanced tryptophan comprising thesteps of: a) transforming a plant cell with the recombinant DNAconstruct of claim 1 or claim 4 for suppression of more than one maizezein target gene; and b) regenerating the plant cell into a fertiletransgenic plant, wherein the plant contains the construct in itsgenome; and c) harvesting seed from the plant, wherein the seed has atleast one of enhanced lysine, enhanced oil and enhanced tryptophanrelative to seed of the same variety not transformed with the construct.10. A method for generating corn seeds having at least one of enhancedlysine, enhanced oil and enhanced tryptophan comprising the steps of: d)transforming a plant cell with the recombinant DNA construct of claim 5or claim 8 for suppression of more than one maize zein target gene; ande) regenerating the plant cell into a fertile transgenic plant, whereinthe plant contains the construct in its genome; and f) harvesting seedfrom the plant, wherein the seed has at least one of enhanced lysine,enhanced oil and enhanced tryptophan relative to seed of the samevariety not transformed with the construct.
 11. A transgenic plantcomprising within its genome a DNA construct of claim 1 or claim 4 forsuppression of more than one maize zein target gene.
 12. A harvestedseed from a plant of claim
 11. 13. A harvested seed from a plant ofclaim 11, wherein the sense-oriented DNA elements are from two genestargeted for suppression and the anti-sense-oriented DNA elements arefrom two genes targeted for suppression.
 14. A processed product of theseed of claim 12, wherein the product is a feed, a meal, or a partiallypurified protein composition.
 15. A transgenic plant comprising withinits genome a DNA construct of claim 5 or claim 8 for suppression of morethan one maize zein target gene.
 16. A harvested seed from a plant ofclaim
 15. 17. A harvested seed from a plant of claim 15, wherein thesense-oriented DNA element is from one gene targeted for suppression andthe anti-sense-oriented DNA elements are from two genes targeted forsuppression.
 18. A processed product of the seed of claim 16, whereinthe product is a feed, a meal, or a partially purified proteincomposition.